Case report: isolation of Hydrogenophaga from septic blood culture following near-death drowning in lakewater

A patient suffered a non-fatal wet drowning in a freshwater lake and developed bacteraemia several days later. Blood culture grew a Gram-negative rod that could not be identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 16S ribosomal RNA sequencing of the isolate identified the microbe as Hydrogenophaga laconesensis – an environmental microbe commonly found in freshwater. The recovery of multiple pathogenic micro-organisms (although not H. laconesensis ) from culture of respiratory specimens prompted the initiation of antibiotic therapy with cefepime and, later, vancomycin. The patient’s clinical course gradually improved over the course of 2 weeks and she was ultimately discharged home with minimal sequelae. To our knowledge, this is the first evidence of human infection with bacteria in the genus Hydrogenophaga . Hydrogenophaga may be considered in cases of freshwater near-drowning, and MALDI-TOF MS databases should be updated to include H. laconesensis .


INTRODUCTION
The genus Hydrogenophaga consists of motile Gram-negative, rod-shaped, yellow-pigmented bacteria commonly found in association with freshwater [1,2].A small number of strains have been studied for their unique enzymatic properties, including the namesake ability to oxidize hydrogen [3,4]).Although viable Hydrogenophaga bacteria are often found in drinking water [2,5], no reports of human infection with bacteria from this genus have been reported.

CASE DESCRIPTION
A previously healthy adolescent female dived into a shallow lake in the Upper Midwest region of the USA during the summer.She failed to resurface and was found floating face down in the water approximately 5 min later.She was minimally responsive upon being pulled from the water.Upon arrival of emergency medical services, fluid was observed in her pharynx and prominent ronchi were heard upon chest auscultation.Blood oxygen saturation was low (80-85 %; reference range: 92-100 %) despite 100 % FiO 2 by bag-valve-mask ventilation.Intubation was attempted twice by ambulance personnel, but was unsuccessful.A supraglottic airway (i-gel, Intersurgical) was placed, but removed shortly after due to aspiration concerns when the patient began vomiting a large volume of liquid consistent with lake water.Following this, a third attempt at intubation by ambulance personnel failed as well and her respirations were again supported with bag-valve-mask ventilation.An aircraft with the regional Helicopter Emergency Medical Service (HEMS) arrived on scene, and HEMS physicians were able to successfully intubate the patient via video laryngoscopy, noting a significant amount of frothy secretions in her airway.She was transferred by helicopter to the University of Wisconsin Hospital.Of note, she was given a full-body chlorhexidine scrub-down upon admission.Radiological evaluation revealed extensive, predominantly centrally located, ground glass and consolidative opacities in both lungs concerning for largevolume aspiration.She was admitted to the intensive care unit (ICU) with a diagnosis of severe acute respiratory distress syndrome (ARDS) secondary to submersion injury.Her PaO 2 /FiO 2 (P/F) ratio on admission was 70 (reference range: normal>=400, severe ARDS <100) despite a positive end-expiratory pressure (PEEP) of 18 cm of water (reference range: 5-10 cm).Approximately 10 h after her drowning incident, the patient became febrile (38.4 °C; reference range: 36.2-37.5 °C) with leukocytosis to 13.4 thousand cells µl −1 (reference range: 3.8-10.5K µl −1 ) and developed progressive vasodilatory shock despite significant doses of norepinephrine (0.3 mcg kg −1 min −1 ) and vasopressin (0.03 units min −1 ).This prompted collection of two blood culture sets (one by venipuncture of the left hand and one via a an existing right internal jugular central venous catheter), sputum and urine cultures, followed by initiation of cefepime for empirical coverage of common freshwater pathogens, including Pseudomonas, Aeromonas and Proteus.

DIAGNOSTIC ASSESSMENT
Culture of respiratory specimens grew Aeromonas sp., Streptococcus pneumoniae, Staphylococcus aureus and endogenous flora at various points throughout the patient's hospital stay.Separately, one of two aerobic blood culture bottles (BACTEC, BD) -the specimen collected from the patient's left hand -flagged as positive on day 2 of blood culture (day 3 of hospitalization) and was subjected to direct-from-specimen matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) (MALDI Biotyper 4.1, Bruker Daltonik, MBT 8468 database).A strong mass spectrum profile was generated for each MALDI-TOF MS attempt, but no high-confidence matches were identified (Fig. 1).Sub-culture yielded a pure culture of non-haemolytic cream-coloured colonies on sheep blood agar after 24 h (Fig. 2a).Growth was also present on chocolate and eosin methylene blue (EMB) agar (Fig. 2b), with Gram stain showing a Gram-negative rod (Fig. 2c).However, this isolate repeatedly failed multiple attempts at identification by MALDI-TOF MS.DNA extraction (Qiagen), 16S rRNA gene real-time PCR (Roche, LC 480), DNA sequencing (Thermo Fisher, ABI 3500xL) and alignment [SmartGene IDNS v3_8_1r1(r31140)] identified a match to Hydrogenophaga laconesensis with 99 % identity in both 5′ and 3′ reads (GenBank accession numbers OP851759-OP851762), forming a contig that contained zero mismatches to reference strain KT756664 across 1444 nucleotides.The concurrent aerobic blood culture (from the right internal jugular) remained negative.Antibiotic susceptibility testing (NM56 panel, MicroScan, Beckman Coulter) on the isolate proved unreliable and was not reported or used for clinical decision-making.

CLINICAL COURSE
The patient's fever, leukocytosis and vasopressor requirement began to improve on cefepime therapy, with the P/F ratio also improving to 200 (i.e.moderate ARDS).On hospital day 3 her antibiotic therapy was narrowed to ceftriaxone.However, her fever returned on hospital day 4, prompting reinitiation of cefepime for an additional 7 day course.Additional blood cultures drawn at this time remained negative.Over the next several days, her fever resolved and her sedation was gradually weaned.She was found to be completely neurologically intact and able to communicate with her care team by writing.Despite her significant overall improvement, her ventilatory and oxygen requirements remained high with progressive bilateral opacities on chest X-ray and multiple failed spontaneous breathing trials (SBTs).Bronchoscopy with bronchoalveolar lavage was performed on hospital day 7 with recovery of endogenous flora and Corynebacterium striatum, prompting initiation of vancomycin therapy.Her respiratory status improved markedly over the next 24 h and she was extubated on hospital day 8, with supplemental oxygen provided by nasal cannula.She was transferred out of the ICU on hospital day 10 and discharged home on room air on hospital day 15 following completion of a 7 day course of vancomycin.Several weeks later, the patient was contacted through the secure messaging portal in her electronic medical record and provided her consent for this case report.A timeline of the patient's clinical course and microbiological workup pertinent to the identification of H. laconesensis can be found in Fig. 3.

DISCUSSION
To our knowledge, this is the first case of human infection with Hydrogenophaga.It seems likely that this organism was present in the lakewater inhaled by the patient given the affinity of Hydrogenophaga species for freshwater and the mechanism of the patient's lung injury.Although H. laconesensis was not recovered from respiratory specimens, this could potentially be explained by low abundance of this organism in the respiratory specimens collected; anatomical variation in specimen collection; out-competition by other bacteria; or mistaking the growth of H. laconesensis colonies for endogenous flora.It is also possible that H. laconesensis was present on this patient's skin and introduced into the blood culture bottle via venipuncture, although the chlorhexidine scrub-down the patient received on admission plus the standard venipuncture site preparation make this less likely.Thus, the route by which H. laconesensis entered the patient's bloodstream; whether this bacteria established a nidus of infection in the lung; and the contribution of pulmonary H. laconesensis infection to ARDS severity, remain open questions.However, the pure culture of H. laconesensis leaves little doubt that bacteraemia could have been caused by a second co-infecting organism.The impact of cefepime and vancomycin therapy on H. laconesensis infection also remains unknown, although the patient did improve while on these antibiotics and subsequent blood cultures were negative.
Three species within the genus Hydrogenophaga are included in the Bruker MALDI-TOF MS MBT 8468 database: H. flava, H intermedia and H. Pseudoflava.MALDI-TOF MS analysis of colonies from the blood agar plate identified H. flava and H. pseudoflava as potential matches (rank #4 and #9, respectively), but log scores were of low confidence (1.290 and 1.180, respectively) (Fig. 1).
In conclusion, Hydrogenophaga infection may be considered in cases of freshwater near-drowning, and updating MALDI-TOF MS databases to include H. laconesensis may be helpful for identification.

Date report received: 08 August 2023 Recommendation: Minor Amendment
Comments: Thank you for submitting your revised manuscript for publication in Access Microbiology.After reviewing the manuscript, I'm happy that all reviewer's comments have been addressed.However, some minor changes are needed to include: 1.For completeness/clarity, please add a statement to the manuscript addressing why co-infection is unlikely and bacteremia was caused by the organism isolated from blood cultures (response to reviewer 1) 2. Line 23: MALDI-TOF MS in full in abstract 3. Hydrogenophaga laconesensis in full for first use in abstract and introduction and then can refer to as H. laconesensis 4. Figure 1

RESPONSE TO REVIEWER COMMENTS
We thank the reviewers for their thoughtful appraisal of our manuscript.In general, reviews were very positive, with the quality of the manuscript rated "Good" (1 of 4 reviewers) or "Very good" (3 of 4 reviewers).The reviewers have pointed out a small number of issues, which we have tried our best to address.Provided below is a point-by-point analysis of their comments, with our response in bold.We believe the changes that we have incorporated improvie the manuscript and make it suitable for publication.
Reviewer #1 1.The authors claim that the infection or bacteraemia is due to the bacterium Hydrogenophaga, but they failed to demonstrate that the infection is strictly due to this species.While the reviewer is correct that a co-infecting microorganism is almost impossible to completely rule out, blood cultures yielded only a single colony morphotype, the purity of which was confirmed by sub-plating, Grams stain, and 16S sequencing.Thus, the most parsimonious explanation is bacteremia caused by the organism that was isolated.
2. So far isolated species of the genus Hydrogenophaga are from fresh water habitats and the chances of being infectious are extremely rare.We agree with the reviewer's assessment, which is why we think this rare case worthy of a case report.
3. Authors should have carried out in vitro (using cell lines) and/or in vivo (using animal models) studies, using the isolated strain, to support their claim.As this is a clinical case report, we feel that this is beyond the scope of the current manuscript.It is also unclear what the reviewer would hope to learn from such studies, and how this would inform the case report as it presently stands.
4. The manuscript is concentrated more on the case description, diagnostic assessment and clinical course than on their main claim of proving that strain Hydrogenophaga is the causative of bacteraemia (especially when they claim that the strain is nonhaemolytic).Thus the present work is too naïve and do not recommend for publication.We respectfully suggest that the current format is appropriate for a case report.

Note that reviewer #2 and #3 had no specific comments
Reviewer #4 1. L22.Surely all "drowning" by definition involves water -so delete "wet".There is actually a medical distinction between "wet" and "dry" drowning, as counterintuitive as this may sound.
L 42. Replace dove with dived.This has been done.on the comments received, it is clear that a major revision of this manuscript will be required before a decision can be made on its publication.I will be pleased to consider a revised manuscript along with a document including a point by point response to each of the reviewers comments.Furthermore, it is important to address the limitations of this study in the discussion as you have not proven that the organism isolated caused the disease.Discussions around future work is also required e.g. in vivo and in vitro studies may be useful to characterise the virulence of the isolate.Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers' comments.I look forward to receiving the revised manuscript.

Reviewer 2 recommendation and comments
https://doi.org/10.1099/acmi.0.000533.v1.3 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.

Anonymous.
Date report received: 05 June 2023 Recommendation: Major Revision

Comments:
The manuscript titled "Case report: isolation of Hydrogenophaga from septic blood culture following near-death drowning in lakewater" by Feichtinger et al. have isolated Hydrogenophaga strain from the blood of a patient who drowned and developed bacteraemia.The manuscript is well written and I complement the authors.Please find my comments below: 1.The authors claim that the infection or bacteraemia is due to the bacterium Hydrogenophaga, but they failed to demonstrate that the infection is strictly due to this species.Further, so far isolated species of the genus Hydrogenophaga are from fresh water habitats and the chances of being infectious are extremely rare.2. Authors should have carried out in vitro (using cell lines) and/or in vivo (using animal models) studies, using the isolated strain, to support their claim.3.
The manuscript is concentrated more on the case description, diagnostic assessment and clinical course than on their main claim of proving that strain Hydrogenophaga is the causative of bacteraemia (especially when they claim that the strain is non-haemolytic).Thus the present work is too naïve and do not recommend for publication.

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Partially support

Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No

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: update figure to either highlight both Hydrogenophaga flava and Hydrogenophaga pseudoflava in the table or have neither highlighted Editor Review Questions Respons This is an open-access article report distributed under the terms of the Creative Commons License.This is an open-access article report distributed under the terms of the Creative Commons License.
L62 Full stop after closing the bracket: start a new sentence.This has been done.L77.Full stop after database:.Start a new sentence.This has been done.L95 Replace the semicolon with a full stop, New sentence starting However….This has been done.Thank you for submitting your manuscript for publication in Access Microbiology.It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology.However, based L65 -69.Split into 2 sentences.This has been done.